Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
  • C12N5/06—Animal cells or tissues; Human cells or tissues
  • C12N5/0602—Vertebrate cells
  • C12N5/0634—Cells from the blood or the immune system
  • C12N5/0636—T lymphocytes
  • C12N5/0637—Immunosuppressive T lymphocytes, e.g. regulatory T cells or Treg
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K40/00—Cellular immunotherapy
  • A61K40/10—Cellular immunotherapy characterised by the cell type used
  • A61K40/11—T-cells, e.g. tumour infiltrating lymphocytes [TIL] or regulatory T [Treg] cells; Lymphokine-activated killer [LAK] cells
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K40/00—Cellular immunotherapy
  • A61K40/20—Cellular immunotherapy characterised by the effect or the function of the cells
  • A61K40/22—Immunosuppressive or immunotolerising
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K40/00—Cellular immunotherapy
  • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
  • A61K40/41—Vertebrate antigens
  • A61K40/416—Antigens related to auto-immune diseases; Preparations to induce self-tolerance
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
  • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
  • C12N15/1138—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against receptors or cell surface proteins
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
  • C12N15/90—Stable introduction of foreign DNA into chromosome
  • C12N15/902—Stable introduction of foreign DNA into chromosome using homologous recombination
  • C12N15/907—Stable introduction of foreign DNA into chromosome using homologous recombination in mammalian cells
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/14—Hydrolases (3)
  • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
  • C12N9/22—Ribonucleases [RNase]; Deoxyribonucleases [DNase]
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K40/00—Cellular immunotherapy
  • A61K40/50—Cellular immunotherapy characterised by the use of allogeneic cells
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2310/00—Structure or type of the nucleic acid
  • C12N2310/10—Type of nucleic acid
  • C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPR]
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
  • C12N2501/20—Cytokines; Chemokines
  • C12N2501/23—Interleukins [IL]
  • C12N2501/2302—Interleukin-2 (IL-2)
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
  • C12N2501/50—Cell markers; Cell surface determinants
  • C12N2501/51—B7 molecules, e.g. CD80, CD86, CD28 (ligand), CD152 (ligand)
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
  • C12N2501/50—Cell markers; Cell surface determinants
  • C12N2501/515—CD3, T-cell receptor complex
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
  • C12N2501/50—Cell markers; Cell surface determinants
  • C12N2501/53—CD2
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2510/00—Genetically modified cells

Definitions

• Treg adoptive cell therapy requires ex vivo expansion of Tregs to generate sufficient numbers of cells.
• the ex vivo expansion needs to be done in such way that the purity, stability, and inherent suppressive functions of Tregs are not compromised.
• Tregs in the first 1-2 weeks of culture and take advantage of the enhanced suppressive activity of Tregs early after activation are provided herein.
• discontinuous stimulation of regulatory T cells can achieve rapid Treg expansion by using alternating periods of stimulation and resting.
• An exemplary DSORTTM process is depicted in Fig. 8.
• expansion rates over 1000-fold such methods can produce a pure, stable, and highly functional Treg product.
• these methods do not require the use of rapamycin to slow down the growth of cells during the first 1-2 weeks of culture.
• the method further comprises genetically engineering the first rested population of Tregs, the second stimulated population of Tregs, the second rested population of Tregs, the third stimulated population of Tregs, the third rested population of Tregs, the further stimulated population of Tregs, or the further rested population of Tregs.
• a method of treating an autoimmune or inflammatory disease in a subject comprising administering to the subject an effective amount a population of Tregs obtained using any method provided herein or any Tregs provided herein.
• a method of treating or preventing graft vs host disease comprising administering to the subject an effective amount a population of Tregs obtained using any method provided herein or any Tregs provided herein.
• the population of Tregs is allogeneic to the subject. In some aspects, the population of Tregs is autologous to the subject.
• Fig. 1 shows the expansion of regulatory T cells (Tregs) subjected to continuous or discontinuous stimulation with either CD3/28/2 tetrameric monoclonal antibodies (mAbs) or Dynabeads. (See Example 2.)
• Fig. 3 shows the expansion of Tregs subjected to continuous stimulation or discontinuous stimulation with an extended non-stimulatory phase. Stimulations were performed with either CD3/28/2 tetrameric monoclonal antibodies (mAbs) or Dynabeads. (See Example 4.)
• polypeptide refers to a protein which includes modifications, such as deletions, additions, and substitutions (generally conservative in nature), to the native sequence, as long as the protein maintains the desired activity. These modifications may be deliberate, as through site-directed mutagenesis, or may be accidental, such as through mutations of hosts which produce the proteins or errors due to PCR amplification.
• variable region typically refers to a portion of an antibody, generally, a portion of a light or heavy chain, typically about the amino-terminal 110 to 120 amino acids or 110 to 125 amino acids in the mature heavy chain and about 90 to 115 amino acids in the mature light chain, which differ extensively in sequence among antibodies and are used in the binding and specificity of a particular antibody for its particular antigen.
• the variability in sequence is concentrated in those regions called complementarity determining regions (CDRs) while the more highly conserved regions in the variable domain are called framework regions (FR).
• CDRs complementarity determining regions
• FR framework regions
• variable region is a human variable region.
• variable region comprises rodent or murine CDRs and human framework regions (FRs).
• FRs human framework regions
• variable region is a primate (e.g, non-human primate) variable region.
• variable region comprises rodent or murine CDRs and primate (e.g, non-human primate) framework regions (FRs).
• determining the absence of a stimulating agent include determining the drownregulation of Treg activation markers (e.g., Foxp3, Helios, CTLA4, CD25, CD69, HLA-DR) and/or determing the downregulation of immunosuppressive cytokine production (e.g. IL-10, TGFb).
• Treg activation markers e.g., Foxp3, Helios, CTLA4, CD25, CD69, HLA-DR
• immunosuppressive cytokine production e.g. IL-10, TGFb
• a resting step there is no stimulatory agent present in a resting step, i.e., the Tregs are cultured without any stimulatory agent.
• Some methods provided herein begin with a resting step.
• a resting step can also occur after a stimulating step and/or can precede a stimulating step. In some aspects, a resting step occurs between two stimulating steps.
• a resting step is about 5 days (about 120 hours).
• a method provided herein comprises a first stimulating step, followed be a first resting step, followed be a second stimulating step, followed by a second resting step, followed by a third stimulating step, followed by a third resting step.
• the Tregs are harvested after a third resting step.
• IL-15 can be utilized in the methods provided herein at a concentration of greater than 0.5 ng/ml. In some aspects, the concentration of IL-15 utilized is more than 1 ng/ml. In some aspects, the concentration of IL-15 utilized is more than 2 ng/ml. In some aspects, the concentration of IL-15 utilized is more than 10 ng/ml. In some aspects, the concentration of IL-15 utilized is more than 50 ng/ml. In some aspects, the concentration ofIL-15 utilized is more than 75 ng/ml. In some aspects, the concentration of IL-15 utilized is more than 100 ng/ml. In some aspects, the concentration of IL-15 utilized is more than 150 ng/ml.
• IL-15 can be utilized in the methods provided at a concentration of greater than 1
• Tregs are cultured in the presence of N-Acetyl-L-cysteine
• Tregs are cultured at a concentration that does not exceed 5 million cells per ml. In some aspects, Tregs are cultured at a concentration that does not exceed 3 million cells per ml. In some aspects, Tregs are cultured at a concentration that does not exceed 2.5 million cells per ml. In some aspects, Tregs are cultured at a concentration that does not exceed 2 million cells per ml. In some aspects, Tregs are cultured at a concentration that does not exceed 1.5 million cells per ml. In some aspects, Tregs are cultured at a concentration that does not exceed 1 million cells per ml.
• the methods provided herein comprise the use of an artificial antigen presenting cell. In some aspects, the methods provided herein do not comprise the use of an artificial antigen presenting cell.
• the Tregs have expanded by about 80-fold by the end of step (b), e.g., by Day 6.
• This antigen engagement stimulates endogenous signal transduction pathways leading to activation and proliferation of the TCR-engineered cells.
• the gene-regulating system can mediate a change in the sequence of an endogenous target gene, for example, by introducing one or more mutations into the endogenous target sequence, such as by insertion or deletion of one or more nucleic acids in the endogenous target sequence.
• exemplary mechanisms that can mediate alterations of the endogenous target sequence include, but are not limited to, non-homologous end joining (NHEJ) (e.g, classical or alternative), microhomology-mediated end joining (MMEJ), homology-directed repair (e.g, endogenous donor template mediated), SDSA (synthesis dependent strand annealing), single strand annealing or single strand invasion.
• the nucleic acid-based gene-regulating system comprises one or more morpholinos.
• “Morpholino” as used herein refers to a modified nucleic acid oligomer wherein standard nucleic acid bases are bound to morpholine rings and are linked through phosphorodiamidate linkages. Similar to siRNA and shRNA, morpholinos bind to complementary mRNA sequences. However, morpholinos function through steric-inhibition of mRNA translation and alteration of mRNA splicing rather than targeting complementary mRNA sequences for degradation.
• the nucleic acid-based gene-regulating system comprises an siRNA molecule or an shRNA molecule selected from those known in the art, such as the siRNA and shRNA constructs available from commercial suppliers such as Sigma Aldrich, Dharmacon, ThermoFisher, and the like.
• the gene-regulating system comprises two or more nucleic acid molecules (e.g, two or more siRNAs, two or more shRNAs, two or more RNA aptamers, or two or more morpholinos), wherein at least one of the nucleic acid molecules binds to a target RNA sequence that is 100% identical to an RNA sequence encoded by a DNA sequence of a target gene selected from Table 1.
• two or more nucleic acid molecules e.g, two or more siRNAs, two or more shRNAs, two or more RNA aptamers, or two or more morpholinos
• Zinc finger-based systems comprise a fusion protein comprising two protein domains: a zinc finger DNA binding domain and an enzymatic domain.
• a “zinc finger DNA binding domain”, “zinc finger protein”, or “ZFP” is a protein, or a domain within a larger protein, that binds DNA in a sequence-specific manner through one or more zinc fingers, which are regions of amino acid sequence within the binding domain whose structure is stabilized through coordination of a zinc ion.
• the zinc finger domain by binding to a target DNA sequence, directs the activity of the enzymatic domain to the vicinity of the sequence and, hence, induces modification of the endogenous target gene in the vicinity of the target sequence.
• a zinc finger domain can be engineered to bind to virtually any desired sequence.
• a zinc finger binding domain comprises one or more zinc fingers.
• the gene-regulating system comprises two or more ZFP-fusion proteins each comprising a zinc finger binding domain, wherein at least one of the zinc finger binding domains binds to a target DNA sequence that is at least 90% identical to a target DNA sequence of a target gene selected from Table 1.
• the gene regulating system comprises two or more ZFP-fusion proteins each comprising a zinc finger binding domain, wherein at least one of the zinc finger binding domains binds to a target DNA sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to a target DNA sequence of a target gene selected from Table 1.
• An exemplary Type IIS restriction enzyme whose cleavage domain is separable from the binding domain, is Fokl. This particular enzyme is active as a dimer. Bitinaite et al. (1998) Proc. Natl. Acad. Sci. USA 95: 10,570-10,575.
• two fusion proteins each comprising a Fokl enzymatic domain, can be used to reconstitute a catalytically active cleavage domain.
• a single polypeptide molecule containing a zinc finger binding domain and two Fokl enzymatic domains can also be used.
• Exemplary ZFPs comprising Fokl enzymatic domains are described in US Patent No. 9,782,437.
• a site-directed modifying polypeptide comprises an activity portion that has enzymatic activity that modifies the endogenous target nucleic acid sequence (e.g., nuclease activity, methyltransferase activity, demethylase activity, DNA repair activity, DNA damage activity, deamination activity, dismutase activity, alkylation activity, depurination activity, oxidation activity, pyrimidine dimer forming activity, integrase activity, transposase activity, recombinase activity, polymerase activity, ligase activity, helicase activity, photolyase activity or glycosylase activity).
• nuclease activity e.g., nuclease activity, methyltransferase activity, demethylase activity, DNA repair activity, DNA damage activity, deamination activity, dismutase activity, alkylation activity, depurination activity, oxidation activity, pyrimidine dimer forming activity, integrase activity, transposa
• the CRISPR/Cas system is a Class 2 and Type VI system, utilizing the Casl3 proteins (e.g., Casl3a (also known as C2c2), Casl3b, and Casl3c).
• Casl3a also known as C2c2
• Casl3b also known as C2c2
• Casl3c the site-directed modifying polypeptide
• the site-directed modifying polypeptide is a Casl3 RNA rib oendonucl ease and the nucleic acid guide molecule is a gRNA.
• Cas9 molecules include Cas9 molecules of a cluster 1 bacterial family, cluster 2 bacterial family, cluster 3 bacterial family, cluster 4 bacterial family, cluster 5 bacterial family, cluster 6 bacterial family, a cluster 7 bacterial family, a cluster 8 bacterial family, a cluster 9 bacterial family, a cluster 10 bacterial family, a cluster 1 1 bacterial family, a cluster 12 bacterial family, a cluster 13 bacterial family, a cluster 14 bacterial family, a cluster 15 bacterial family, a cluster 16 bacterial family, a cluster 17 bacterial family, a cluster 18 bacterial family, a cluster 19 bacterial family, a cluster 20 bacterial family, a cluster 21 bacterial family, a cluster 22 bacterial family, a cluster 23 bacterial family, a cluster 24 bacterial family, a
• the Cas polypeptides described herein can be engineered to alter the PAM/PFS specificity of the Cas polypeptide.
• a mutant Cas polypeptide has a PAM/PFS specificity that is different from the PAM/PFS specificity of the parental Cas polypeptide.
• a naturally occurring Cas protein can be modified to alter the PAM/PFS sequence that the mutant Cas polypeptide recognizes to decrease off target sites, improve specificity, or eliminate a PAM/PFS recognition requirement.
• a Cas protein can be modified to increase the length of the PAM/PFS recognition sequence.
• the length of the PAM recognition sequence is at least 4, 5, 6, 7, 8, 9, 10 or 15 amino acids in length.
• the present disclosure provides guide RNAs (gRNAs) that direct a site-directed modifying polypeptide to a specific target nucleic acid sequence.
• a gRNA comprises a nucleic acid-targeting segment and protein-binding segment.
• the nucleic acid-targeting segment of a gRNA comprises a nucleotide sequence that is complementary to a sequence in the target nucleic acid sequence.
• the nucleic acid-targeting segment of a gRNA interacts with a target nucleic acid in a sequence-specific manner via hybridization (i.e., base pairing), and the nucleotide sequence of the nucleic acid-targeting segment determines the location within the target nucleic acid that the gRNA will bind.
• the nucleic acid-targeting segment of a gRNA can be modified ( e.g ., by genetic engineering) to hybridize to any desired sequence within a target nucleic acid sequence.
• the gene-regulating system comprises two or more gRNA molecules each comprising a DNA-binding segment, wherein at least one of the nucleic acid-binding segments binds to a target DNA sequence that is at least 90% identical to a target DNA sequence of a target gene selected from Table 1.
• the gene regulating system comprises two or more gRNA molecules each comprising a nucleic acid-binding segment, wherein at least one of the nucleic acid-binding segments binds to a target DNA sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to a target DNA sequence of a target gene selected from Table 1.
• Tregs are genetically engineered after at least 6 days in culture.
• Tregs are genetically engineered after about 6 days in culture. In some aspects, Tregs are genetically engineered after about 7 days in culture. In some aspects, Tregs are genetically engineered after about 8 days in culture. In some aspects, Tregs are genetically engineered after about 9 days in culture. In some aspects, Tregs are genetically engineered after about 10 days in culture.
• Tregs are isolated by enriching CD25+ T cells using magnetic beads. In some aspects, Tregs are isolated by enriching CD25+ T cells using Fluorescent Activated Cell Sorting (FACS).
• FACS Fluorescent Activated Cell Sorting
• gRNAs where indicated, all experiments use single-molecule gRNAs (sgRNAs).
• Dual gRNA molecules were formed by duplexing 200 mM tracrRNA (IDT Cat# 1072534) with 200 pM of target-specific crRNA (IDT) in nuclease free duplex buffer (IDT Cat#l 1- 01-03-01) for 5 min at 95° C, to form 100 mM of tracrRNAxrRNA duplex, where the tracrRNA and crRNA are present at a 1:1 ratio.
• nucleofection buffer 18% supplement 1, 82% P3 buffer from the Amaxa P3 primary cell 4D- Nuclefector X kit S (Cat # V4XP-3032)
• This cell suspension was combined with the RNP solution form the step above and an inert single-stranded DNA oligonucleotide (Alt-R Cas9 Electroporation Enhancer) at a ratio of 20:5:1.
• Cells were electroporated following the “T cell, Human, Stim” program (EO-115).
• X-VIVO 15 media After electroporation, 80 pL of warm X-VIVO 15 media was added to each well, and cells were pooled into a culture flask at a density of 2 x 10 6 cells/mL in X-VIVO 15 media containing IL-2 (300 units/mL).
• genomic DNA was isolated from edited T cells using the Qiagen Blood and Cell Culture DNA Mini Kit (Cat #: 13323) following the vendor recommended protocol and quantified.
• PCR was performed to amplify the region of edited genomic DNA using locus- specific PCR primers containing overhangs required for the addition of Illumina Next Generation sequencing adapters.
• the resulting PCR product was run on a 1% agarose gel to ensure specific and adequate amplification of the genomic locus occurred before PCR cleanup was conducted according to the vendor recommended protocol using the Monarch PCR & DNA Cleanup Kit (Cat#: T1030S).
• Human Tregs were FACS sorted as O ⁇ 45K + O ⁇ 25 w O ⁇ 127 10 from CD4+ T cells isolated from peripheral blood and then stimulated with either CD3/28/2 tetrameric antibody (open symbols) or CD3/28 coated magnetic Dynabeads in the presence of IL-2 and NAC. After 3 days of culture, the cells were washed, counted, and then divided into two separate wells containing media and IL-2 with or without tetrameric antibody (Ab)/Dynabeads. At day 6 of culture, cells were counted again, at which point discontinuously stimulated Tregs had grown ⁇ 40-fold as compared to only ⁇ 20-fold for continuously stimulated Tregs. (See Figure 1.) The difference in growth between the two conditions was observed regardless of whether the cells were stimulated with tetrameric Ab or Dynabeads. (See id.)
• Tregs The number of viable Tregs was determined at the day of transfection, one day post-transfection, and then every 1-2 days for the remainder of the study. While DSORTTM Tregs (open squares) continued to grow another 3-fold following engineering, standard Tregs (closed circles) failed to recover to pre-engineering levels after transfection. (See Figure 4.)
• Helios is a transcription factor that reinforces the expression of Foxp3 in Tregs.
• Tregs that maintain stability from one cellular generation to the next have a fully demethylated Treg-specific demethylated region (TSDR, a locus within the Foxp3 gene), whereas the TSDR of Tregs that have converted from effector T cells in vitro (so-called induced Tregs) or are prone to destabilize under inflammatory conditions is partially methylated.
• the Tregs in this experiment were sorted into four subsets based on their expression of Foxp3 and Helios. Sorted cells were then analyzed using a DNA methylation assay (Pyrosequencing) to determine the level of methylation at the TSDR locus. The results, shown in Figure 6A, demonstrate that only Helios+ Tregs had fully demethylated TSDRs.

Landscapes

• Health & Medical Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Engineering & Computer Science (AREA)
• Genetics & Genomics (AREA)
• Biomedical Technology (AREA)
• Chemical & Material Sciences (AREA)
• Organic Chemistry (AREA)
• Bioinformatics & Cheminformatics (AREA)
• General Health & Medical Sciences (AREA)
• Zoology (AREA)
• Wood Science & Technology (AREA)
• Biotechnology (AREA)
• General Engineering & Computer Science (AREA)
• Molecular Biology (AREA)
• Immunology (AREA)
• Public Health (AREA)
• Veterinary Medicine (AREA)
• Animal Behavior & Ethology (AREA)
• Microbiology (AREA)
• Biochemistry (AREA)
• Epidemiology (AREA)
• Biophysics (AREA)
• Plant Pathology (AREA)
• Physics & Mathematics (AREA)
• Cell Biology (AREA)
• Medicinal Chemistry (AREA)
• Hematology (AREA)
• Mycology (AREA)
• Chemical Kinetics & Catalysis (AREA)
• General Chemical & Material Sciences (AREA)
• Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
• Pharmacology & Pharmacy (AREA)
• Micro-Organisms Or Cultivation Processes Thereof (AREA)
• Medicines Containing Material From Animals Or Micro-Organisms (AREA)
• Peptides Or Proteins (AREA)

Priority Applications (6)

Applications Claiming Priority (2)

Publications (1)

Family

ID=83048425

Family Applications (1)

Country Status (7)

Cited By (2)

Citations (5)

Family Cites Families (7)

• 2022
  • 2022-02-23 CN CN202280016734.1A patent/CN116897201A/zh active Pending
  • 2022-02-23 CA CA3206049A patent/CA3206049A1/en active Pending
  • 2022-02-23 WO PCT/US2022/017526 patent/WO2022182763A1/en not_active Ceased
  • 2022-02-23 AU AU2022226163A patent/AU2022226163A1/en active Pending
  • 2022-02-23 JP JP2023550647A patent/JP2024507373A/ja active Pending
  • 2022-02-23 US US18/547,627 patent/US20240228962A9/en active Pending
  • 2022-02-23 EP EP22760340.4A patent/EP4297757A4/en active Pending

Patent Citations (5)

Non-Patent Citations (1)

Also Published As

Similar Documents

Legal Events